The Mechanism Of SiCEP3 From Setaria Italica In Inhibiting Primary Root Growth Of Plants

Plant small peptide hormones are short active polypeptides formed from their precursor proteins by specific proteolysis and amino acid modification.They act as important signal molecules for intercellular signal transduction and play an important roles in plant growth,development and various stress responses.The CEP（C-terminally encoded peptide）small peptides can be bound by the plama memebrane located receptor CEPRs,triggering intracellular signal transduction,and then regulating downstream pathways.Recent studies have found that CEPs inhibit the growth and development of plants,but the underlying mechanism is still unclear.Foxtail millet（Setaria italica）is an important food crop and a new type of C4 model crop.The unclear mechanism about its growth,development and stress responses limit its production.In this study,the underlying mechanism of small peptide hormone CEP-regulated primary root growth was explored.The relationship between CEP and its receptor activity,and the uptake,transport and signaling of the plant hormone abscisic acid（ABA）were revealed.These results provided a new information for further uncovering CEP regulated plant growth and abiotic stress response.The main results are as follows:（1）Identification of the gene encoding the small CEP peptide in foxtail millet.Through sequence alignment analysis of CEP domain,14 SiCEPs genes were identified from foxtail millet genome.Amino acid cluster analysis showed that the precursor proteins of SiCEPs were divided into two subfamilies.Protein structure analysis exhibited that SiCEPs contain a Nterminal signal peptide and a conserved C-terminal CEP domain,indicating that SiCEPs may have functional redundancy and CEP domain is critical for its function.（2）Tissue expression patterns of SiCEPs were analyzed.RT-qPCR was used to detect the expression of SiCEPs in foxtail millet roots,stems,leaves and panicles.The results showed that all SiCEPs genes were highly expressed in roots;SiCEP8 and SiCEP9 genes were also highly expressed in stems and leaves;one of the gene clusters SiCEP3,SiCEP4 and SiCEP5 were highly expressed in panicles.These results suggested that different SiCEPs may be involved in the development of different organs of foxtail millet.（3）The abiotic stress-induced expression patterns of SiCEPs were analyzed.Firstly,the cis-elements of SiCEPs promoter were analyzed by website prediction,and it was found that their promoters contained phytohormones and abiotic stress-related response elements.Then RT-qPCR was used to detect the expression level of SiCEPs under salt,ABA,and PEG treatments.The results showed that the expression of SiCEPs were mainly induced by osmotic stress,salinity,and ABA treatment.The inducible expression of SiCEP3/4/5 genes was the most obvious.（4）SiCEP3 promotes ABA import and transport.We found that exogenous application of mature SiCEP3 inhibited foxtail millet primary root growth,which is similar to the effect of ABA on the roots.We further applied different concentrations of SiCEP3 in the presence of ABA.The results showed that with the increasing concentrations of SiCEP3,the inhibitory effect of ABA on the primary root growth of the foxtail millet seedling was aggravated,and the ABA content also gradually increased,indicating that SiCEP3 may promote ABA import and transport.The results showed that the growth of the overexpression SiCEP3 transgenic hairy roots were significantly inhibited.In the presence of ABA,the ABA content in the overexpression SiCEP3 hairy roots were significantly increased,further confirmed that SiCEP3 promotes ABA uptake and transport.（5）SiCEP3 activates the expression of genes involving in ABA uptake,transport and signaling.The expression of genes encoding ABA uptake,transport and signaling was detected by RT-qPCR under SiCEP3,ABA and combination treatments.The results showed that the exogenous application of mature SiCEP3 induced the expression of genes coding for ABA transporters and receptors.Their expression levels were further increased after superimposing ABA.The expression of genes coding for ABA uptake,transport and signaling was also elevated in the hairy roots of foxtail millet overexpressing SiCEP3.This indicated that SiCEP3 activated ABA uptake,transport and signaling at the transcriptional level,thereby inhibiting foxtail millet primary root growth.（6）SiCEP3 also inhibits the primary root growth in Arabidopsis.To further explore the mechanism of SiCEP3,we treated Arabidopsis wild type and ABA-related mutants with SiCEP3 and ABA alone or in combination.The results showed that in the presence of ABA,the primary root of Arabidopsis thaliana seedlings became shorter,and the ABA content in the seedlings gradually increased with the increasing concentrations of SiCEP3.However,the inhibitory effect on the primary root of nrt1.2abcg40 and pyr1pyl124 mutants were weakened,and the ABA content did not increase.The inhibitory effect on the primary root growth of cyp707a1 and cyp707a2 mutants were aggravated,and the ABA content was higher than wild type,the expression levels of AtABCG40 and AtPYL1/2/3/4 were significantly higher under the combination treatment of SiCEP3 and ABA than alone.These data indicate that the function of SiCEP3 is conserved in Arabidopsis and can also inhibit Arabidopsis primary root growth by promoting ABA uptake,transport and signaling.（7）SiCEP3 and ABA inhibit the kinase activities of the CEP receptor CEPRs.CEPR is the receptor kinase of CEP.To study how SiCEP3 acts through its receptors,we determined the kinase activities of CEPR1 and CEPR2,and found that SiCEP3 and ABA inhibited the kinase activities of AtCEPR1 and AtCEPR2 in in vitro kinase assays.